Rescue of shRNA–knockdown with shRNA–resistant dcDNA.

CD-1 mice were transfected at P0 with Ub-GFP and shRNA against Gapdh, Lman1 or Wisp1 by sub-retinal injection and in vivo electroporation. shRNA-resistant degenerate cDNA (dcDNA) was co-injected together with shRNA against Gapdh, Lman1 or Wisp1 for rescue experiments. Retinas were harvested at P20 and examined for GFP fluorescence (green), Rho immuno-reactivity (red) and DAPI staining (blue). Three biological replicate retinas were collected and imaged. (A, C). Scale bar: 20 µM. (B, D) Higher magnification images of (A, C). OS: outer segment. OONL: outer portion of the outer nuclear layer. IONL: inner portion of the outer nuclear layer. Scale bar: 10 µM. GFP positive (+) cells were counted in sections of retina electroporated with shRNA targeting Gapdh or NRL target genes (E, H). Distribution of electroporated cell bodies in the retina (F). Fraction of GFP positive cells in the retinal outer nuclear layer is counted. OONL, outer region of outer nuclear layer; IONL, inner region of outer nuclear layer. Average outer segment (OS) lengths of electroporated cells were measured (G). Data are represented as mean ± SD. (E–H) *P<0.01, **P<0.001 by Student's t test (n = 3 electroporated retinas).